This invention relates generally to cancer diagnosis and therapy, and more specifically, to cancers associated with over- or underexpression of Bin1 or other members of the BAR family of adaptor proteins.
Bin1/Amphiphysin/RVS (BAR) proteins are a family of adaptor proteins implicated in a diverse set of cellular processes, including tumnorigenesis, cell survival, differentiation, and nerve synaptic activity. BAR proteins share a common N-terminal BAR domain also termed the RVS domain. While BAR proteins share a common domain (BAR), they appear to have divergent physiological functions. As one example, amphiphysin is a neuronal protein of this family which is implicated in synaptic vesicle endocytosis [Wigge and McMahon, Trends Neurosci. 21: 339-344 (1998)]. Amphiphysin is also a paraneoplastic autoimmune antigen in cancers of the breast, lung, and other tissues [Antoine et al, Arch. Neurol. 56: 172-177 (1999); Dropcho, Ann. Neurol. 39: 659-667(1996); Folli et al., N. Engl. J. Med. 328: 546-51 (1993)].
Bin1 (Bridging INtegrator-1) is a second, ubiquitous BAR protein that was initially identified in mammalian cells through its ability to interact with and inhibit the oncogenic properties of c-Myc [Sakarnuro et al., Nature Genet. 14: 69-77 (1996)]. Ubiquitous Bin1 isoforms that localize to the nucleus have tumor suppressor properties and have been implicated in growth control, differentiation, and programmed cell death [Mao et al., Genomics 56: 51-58 (1999); Prendergast, Oncogene 18: 2966-2986 (1999); Sakamuro et al. 1996, cited above; Wechsler-Reya et al., Mol. Cell. Biol. 18: 566-575 (1998)].
Other members of the BAR family include the yeast proteins RVS 167 and RVS161, which are believed to have some negative role in cell growth regulation. There exists a need in the art for compositions and methods useful for diagnosis and treatment of conditions characterized by inappropriate cell growth control, or disorders affecting cell survival, differentiation, endocytosis, and actin organization.
The present invention provides a novel member of the Bin1/Amphiphysin/RVS (BAR) proteins, termed herein Bin2. Bin2 proteins, nucleic acids, and other Bin2 compositions of the invention have a variety of uses related to regulation of cell growth control, cell survival, differentiation, endocytosis and actin organization, as well as for the diagnosis and treatment of conditions associated with aberrant cell behavior.
In one aspect, the present invention provides a Bin2 protein. In one desirable embodiment, the protein has the 564 amino acid sequence of SEQ ID NO:2. In another embodiment, the present invention provides a Bin2 peptide or protein selected from the group consisting of a fragment of Bin2 comprising at least 8 amino acids in length. In one embodiment a fragment of this invention is at least 8 contiguous amino acids in length and is selected from amino acids 1 to 13 of SEQ ID NO:2. In another embodiment, a fragment of this invention is at least 14 amino acids in length and includes amino acids 23 to 35 of SEQ ID NO:2, and preferably amino acids 2345 of SEQ ID NO: 2. In still another embodiment, a fragment includes amino acids 138-155 of SEQ ID NO:2 and comprises at least 19 amino acids in length. In still another embodiment, a fragment includes amino acids 179-336, or a smaller fragment of at least 8 amino acids contained therein. Still other fragments may be selected from the sequence. In yet another embodiment, the invention provides analogs or homologs of SEQ ID NO:2. In still another embodiment, the invention provides a fusion protein comprising the amino acid sequence of SEQ ID NO: 2, a fragment, analog or homolog thereof, and a fusion partner. In still a further embodiment, the invention provides a deletion protein comprising the amino acid sequence of SEQ ID NO:2 with one to twenty amino acids deleted therefrom.
In another aspect, the present invention provides a Bin2 nucleic acid sequence. Desirably, the Bin2 nucleic acid sequence encodes a protein or fragment of the invention (such as those mentioned above) and contains SEQ ID NO:1 or a fragment thereof. In one embodiment, the Bin2 nucleic acid sequence hybridizes to the sequence of SEQ ID NO:1 under stringent conditions. In another embodiment, the invention provides a nucleic acid sequence complementary to the nucleic acid sequence of SEQ ID NO:1. In still another embodiment, the invention provides a nucleic acid sequence encoding a fusion protein of the invention. In a further embodiment, the invention provides an allelic variant of any of the Bin2 nucleic acid sequences of the invention. In still another embodiment, the nucleic acid sequence is an antisense sequence to the sequences described above.
In a further aspect, the invention provides a vector comprising a Bin2 nucleic acid sequence of the invention under the control of regulatory sequences which direct expression of the Bin2 protein.
In still another aspect, the invention provides a host cell transformed with the vector of the invention.
In yet a further aspect, the invention provides a diagnostic reagent comprising a Bin2 nucleic acid sequence of the invention and a detectable label which is associated with said sequence. Methods of diagnosing conditions associated with inappropriate functional levels, the loss of expression of Bin2 or altered expression of Bin2, e.g., cancers, which use this reagent are also provided.
In still a further aspect, the invention provides a diagnostic reagent comprising a Bin2 protein or peptide of the invention and a detectable label which is associated with that protein. Also provides are methods of using this reagent and/or the Bin2 protein for diagnosing cancers associated with inappropriate expression (e.g., overexpression or underexpression or altered expression) of Bin1, to which Bin2 binds. This method involves the steps of contacting a sample from a human or animal to be diagnosed with the Bin2 protein of the invention, or the diagnostic reagent containing this protein, whereby in the presence of Bin1 in the sample, a complex is formed between Bin1 and the Bin2 protein or reagent, and analyzing for the presence of said complex.
In yet another aspect, the invention provides an isolated anti-Bin2 antibody which is specific for the Bin2 protein of the invention.
In still another aspect, the invention provides a diagnostic reagent comprising the anti-Bin2 antibody of the invention and a detectable label. Further provided by the invention is a method of diagnosing cancer or hyperplastic disease characterized by inappropriate levels or altered expression of functional Bia2 in a human or an animal using the anti-Bin2 antibody or diagnostic reagent of the invention. This method involves contacting an anti-Bin2 antibody or a diagnostic reagent containing same with a sample from a human or animal to be diagnosed, whereby in the presence of Bin2, a detectable complex is formed with the Bin2 protein or diagnostic reagent, analyzing for the presence or absence of said complex; and comparing the level of complex to a standard, wherein the absence of said detectable label indicates the absence of functional Bin2.
In still another aspect, the invention provides a kit for diagnosing a condition associated with Bin2 comprising a diagnostic reagent of the invention.
In a further aspect, the invention provides an anti-idiotype antibody specific for the anti-Bin2 antibody of the invention.
In yet another aspect, the invention provides a composition comprising an effective amount of a Bin2 protein or anti-idiotype of the invention and a pharmaceutically acceptable carrier.
In still another aspect, the invention provides a method of detecting inappropriate expression of Box dependent myc-interacting protein-2 (Bin2) in a patient comprising providing a sample from a patient suspected of having said inappropriate (over- or under-expression) or altered expression; incubating said sample in the presence of an anti-Bin2 antibody or a diagnostic reagent containing same; and comparing levels of expression to a normal Bin2 control.
In a further aspect, the invention provides a method of detecting inappropriate expression of Box-dependent myc-interacting peptide-2 Bin2) in a patient comprising providing a sample from a patent suspected of having said inappropriate expression and performing nucleic acid amplification using a Bin2 nucleic acid sequence of the invention.
In still a further aspect, the invention provides a method of identifying compounds which specifically bind to Bin2 or which specifically inhibit or block the binding of Bin2 to its ligand. In one embodiment, the method involves comprising the steps of contacting said Bin2 or a fragment thereof with a test compound to permit binding of the test compound to Bin2; and determining the amount of test compound which is bound to Bin2. In another embodiment, the invention provides a method of contacting an amount of immobilized first Bin peptide or a fragment thereof with a test compound and an amount of labeled second Bin peptide or fragment, wherein said first Bin peptide is either a Bin1 or a Bin2 peptide, and the second Bin peptide is the Bin peptide that binds said first Bin peptide. Unbound material is separated from immobilized material and the amount of label on said immobilized material is measured. A decrease in the amount of label immobilized in the presence of test compound compared to the amount of label immobilized in the presence of a control peptide or protein, indicates that said test compound inhibits the binding of Bin1 to Bin 2, or vice versa. The invention further provides a compound identified by this method.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.